黒尾 誠

BACKGROUND:   Gastrointestinal symptoms, particularly constipation, increase with aging, but their underlying mechanisms are poorly understood due to lack of experimental models. Previously we established the progeric klotho mouse as a model of aging-associated anorexia and gastric dysmotility. We also detected reduced fecal output in these animals; therefore, the aim of this study was to investigate in vivo function and cellular make-up of the small intestinal and colonic neuromuscular apparatus. METHODS: Klotho expression was studied by RT-PCR and immunohistochemistry. Motility was assessed by dye transit and bead expulsion. Smooth muscle and neuron-specific gene expression was studied by Western immunoblotting. Interstitial cells of Cajal (ICC) and precursors were analyzed by flow cytometry, confocal microscopy, and three-dimensional reconstruction. HuC/D(+) myenteric neurons were enumerated by fluorescent microscopy. KEY RESULTS: Klotho protein was detected in neurons, smooth muscle cells, and some ICC classes. Small intestinal transit was slower but whole-gut transit of klotho mice was accelerated due to faster colonic transit and shorter intestinal lengths, apparent only after weaning. Fecal water content remained normal despite reduced output. Smooth muscle myosin expression was reduced. ICC, ICC precursors, as well as nitrergic and cholinergic neurons maintained their normal proportions in the shorter intestines. CONCLUSIONS & INFERENCES: Progeric klotho mice express less contractile proteins and develop generalized intestinal neuromuscular hypoplasia mainly arising from stunted postweaning growth. As reduced fecal output in these mice occurs in the presence of accelerated colonic and whole-gut transit, it likely reflects reduced food intake rather than intestinal dysmotility.

Klotho is a putative aging suppressor gene encoding a single-pass transmembrane co-receptor that makes the fibroblast growth factor (FGF) receptor specific for FGF-23. In addition to multiple endocrine organs, Klotho is expressed in kidney distal convoluted tubules and parathyroid cells, mediating the role of FGF-23 in bone-kidney-parathyroid control of phosphate and calcium. Klotho(-/-)mice display premature aging and chronic kidney disease-associated mineral and bone disorder (CKD-MBD)-like phenotypes mediated by hyperphosphatemia and remediated by phosphate-lowering interventions (diets low in phosphate or vitamin D; knockouts of 1α-hydroxylase, vitamin D receptor, or NaPi cotransporter). CKD can be seen as a state of hyperphosphatemia-induced accelerated aging associated with Klotho deficiency. Humans with CKD experience decreased Klotho expression as early as stage 1 CKD; Klotho continues to decline as CKD progresses, causing FGF-23 resistance and provoking large FGF-23 and parathyroid hormone increases, and hypovitaminosis D. Secreted Klotho protein, formed by extracellular clipping, exerts FGF-23-independent phosphaturic and calcium-conserving effects through its paracrine action on the proximal and distal tubules, respectively. We contend that decreased Klotho expression is the earliest biomarker of CKD and the initiator of CKD-MBD pathophysiology. Maintaining normal phosphate levels with phosphate binders in patients with CKD with declining Klotho expression is expected to reduce mineral and vascular derangements.

Klotho is a single-pass transmembrane protein that exerts its biological functions through multiple modes. Membrane-bound Klotho acts as coreceptor for the major phosphatonin fibroblast growth factor-23 (FGF23), while soluble Klotho functions as an endocrine substance. In addition to in the distal nephron where it is abundantly expressed, Klotho is present in the proximal tubule lumen where it inhibits renal Pi excretion by modulating Na-coupled Pi transporters via enzymatic glycan modification of the transporter proteins - an effect completely independent of its role as the FGF23 coreceptor. Acute kidney injury (AKI) and chronic kidney disease (CKD) are states of systemic Klotho deficiency, making Klotho a very sensitive biomarker of impaired renal function. In addition to its role as a marker, Klotho also plays pathogenic roles in renal disease. Klotho deficiency exacerbates decreases in, while Klotho repletion or excess preserves, glomerular filtration rate in both AKI and CKD. Soft tissue calcification, and especially vascular calcification, is a dire complication in CKD, associated with high mortality. Klotho protects against soft tissue calcification via at least 3 mechanisms: phosphaturia, preservation of renal function and a direct effect on vascular smooth muscle cells by inhibiting phosphate uptake and dedifferentiation. In summary, Klotho is a critical molecule in a wide variety of renal diseases and bears great potential as a diagnostic and prognostic biomarker as well as for therapeutic replacement therapy.

Although the tandem duplication of mitochondrial (mt) sequences, especially those of the control region (CR), has been detected in metazoan species, few studies have focused on the features of the duplicated sequence itself, such as the gene conversion rate, distribution patterns of the variation, and relative rates of evolution between the copies. To investigate the features of duplicated mt sequences, we partially sequenced the mt genome of 16 Phoebastria albatrosses belonging to three species (P. albatrus, P. nigripes, and P. immutabilis). More than 2,300 base pairs of tandemly-duplicated sequence were shared by all three species. The observed gene arrangement was shared in the three Phoebastria albatrosses and suggests that the duplication event occurred in the common ancestor of the three species. Most of the copies in each individual were identical or nearly identical, and were maintained through frequent gene conversions. By contrast, portions of CR domains I and III had different phylogenetic signals, suggesting that gene conversion had not occurred in those sections after the speciation of the three species. Several lines of data, including the heterogeneity of the rate of molecular evolution, nucleotide differences, and putative secondary structures, suggests that the two sequences in CR domain I are maintained through selection; however, additional studies into the mechanisms of gene conversion and mtDNA synthesis are required to confirm this hypothesis.

Klotho is a transmembrane protein that can be shed and act as a circulating hormone and is a putative tumor suppressor in breast cancer. A functional variant of KLOTHO (KL-VS) contains two amino acid substitutions F352V and C370S and shows reduced activity. Germ-line mutations in BRCA1 and BRCA2 substantially increase lifetime risk of breast and ovarian cancers. Yet, penetrance of deleterious BRCA1 and BRCA2 mutations is incomplete even among carriers of identical mutations. We examined the association between KL-VS and cancer risk among 1115 Ashkenazi Jewish women: 236 non-carriers, 631 BRCA1 (185delAG, 5382insC) carriers and 248 BRCA2 (6174delT) carriers. Among BRCA1 carriers, heterozygosity for the KL-VS allele was associated with increased breast and ovarian cancer risk (hazard ratio 1.40, 95% confidence intervals 1.08-1.83, P=0.01) and younger age at breast cancer diagnosis (median age 48 vs 43 P=0.04). KLOTHO and BRCA2 are located on 13q12, and we identified linkage disequilibrium between KL-VS and BRCA2 6174delT mutation. Studies in breast cancer cells showed reduced growth inhibitory activity and reduced secretion of klotho F352V compared with wild-type klotho. These data suggest KL-VS as a breast and ovarian cancer risk modifier among BRCA1 mutation carriers. If validated in additional cohorts, the presence of KL-VS may serve as a predictor of cancer risk among BRCA1 mutation carriers.

Chromosomes stained with fluorochromes, including quinacrine mustard (QM), emit the brightest fluorescence immediately after exposure to excitation light, and the fluorescence gradually fades with an increase in exposure time. However, in the QM-stained chromosomes of the small Japanese field mouse Apodemus argenteus, most C-heterochromatic regions emit weak fluorescence immediately after exposure to blue light, and they become brightly fluorescent by prolonged exposure (delayed QM fluorescence). We proposed recently that the delayed QM fluorescence is somehow related to nicks produced in C-heterochromatic DNA by blue light irradiation. To test this possibility, we examined the chromosomal distribution of nicks by in-situ nick translation and changes, if any, in the QM fluorescence pattern after methylene blue (MB) -mediated photooxidation, which is considered to induce nicks in chromosomal DNA. It was found that C-heterochromatic regions fluoresced brightly without any delay after exposure to blue light, and that nicks increased considerably in the same regions after the MB-mediated photooxidation. It seems, therefore, that photooxidation and strand breaks in DNA (including nicks) are responsible for the induction of delayed QM fluorescence. Trypsin digestion, on the other hand, abolished delayed QM fluorescence. Thus, not only DNA but also chromosomal protein(s) are involved in the unusual sequence of QM fluorescence patterns in A. argenteus.

Endocrine fibroblast growth factors (FGFs) function as hormones that maintain specific metabolic states by controlling homeostasis of bile acid, glucose, fatty acid, phosphate, and vitamin D. Endocrine FGFs exert their biological activity through a common design of coreceptor system consisting of the Klotho gene family of transmembrane proteins and cognate FGF receptors. Moreover, expression of endocrine FGFs is regulated by nuclear receptors whose lipophilic ligands are generated under the control of these hormones in the target organs. Thus, novel endocrine axes have emerged that regulate divers e metabolic processes through feedback loops composed of the FGF, Klotho, FGF receptor, and nuclear receptor gene families. This review summarizes the role of Klotho family proteins in the regulation of metabolic activity and expression of the endocrine FGFs. (C) 2009 International Union of Biochemistry and Molecular Biology, Inc. Volume 35, Number 1, January/February 2009, Pages 52-60. E-mail: makoto.kuro-o@utsouthwestern.edu

Klotho is an anti-aging gene, which has been shown to inhibit the insulin and insulin-like growth factor 1 (IGF-1) pathways in mice hepatocytes and myocytes. As IGF-1 and insulin regulate proliferation, survival and metastasis of breast cancer, we studied klotho expression and activities in human breast cancer. Immunohistochemistry analysis of klotho expression in breast tissue arrays revealed high klotho expression in normal breast samples, but very low expression in breast cancer. In cancer samples, high klotho expression was associated with smaller tumor size and reduced KI67 staining. Forced expression of klotho reduced proliferation of MCF-7 and MDA-MB-231 breast cancer cells, whereas klotho silencing in MCF-7 cells, which normally express klotho, enhanced proliferation. Moreover, forced expression of klotho in these cells, or treatment with soluble klotho, inhibited the activation of IGF-1 and insulin pathways, and induced upregulation of the transcription factor CCAAT/enhancer-binding protein beta, a breast cancer growth inhibitor that is negatively regulated by the IGF-1-AKT axis. Co-immunoprecipitation revealed an interaction between klotho and the IGF-1 receptor. Klotho is also a known modulator of the fibroblast growth factor (FGF) pathway, a pathway that inhibits proliferation of breast cancer cells. Studies in breast cancer cells revealed increased activation of the FGF pathway by basic FGF following klotho overexpression. Klotho did not affect activation of the epidermal growth factor pathway in breast cancer cells. These data suggest klotho as a potential tumor suppressor and identify it as an inhibitor of the IGF-1 pathway and activator of the FGF pathway in human breast cancer.

The aim of this study was to determine the prevalence of Cryptosporidium in snakes in Japan. Fecal samples or intestinal contents of 469 snakes, consisting of five species, were analyzed and Cryptosporidium oocysts were detected only from the Japanese grass snake Rhabdophis tigrinus. The mean prevalence of Cryptosporidium sp. in Japanese grass snakes was approximately 26% in the region studied. Histopathological observations revealed that the organism caused proliferative enteritis in the small intestine. Sequence analysis of a fragment of the small subunit rRNA gene has shown that the partial sequence of Cryptosporidium sp. isolated from the snakes was identical to that of the Cryptosporidium snake genotype W11 from New Guinea viper boa.

"Delayed QM-fluorescence" refers to the unusual kinetics of fluorescence from most of the C-heterochromatic regions of the chromosomes of the small Japanese field mouse Apodemus argenteus. When stained with quinacrine mustard (QM-stained), these C-heterochromatic regions emit weak fluorescence immediately after exposure to blue light (BL); they emit bright fluorescence within a few minutes; and the intensity of the fluorescence gradually decreases after maximum fluorescence has been recorded. To elucidate the mechanism of this phenomenon, we used acridine orange staining (AO-staining) and a modified version of the in situ nick-translation method. Focusing on the large C-heterochromatic region (C-block) of the X chromosome, we noted that AO-stained C-blocks emitted greenish fluorescence, while QM-stained and BL-exposed (QM-BL-processed) C-blocks emitted reddish fluorescence upon AO-staining after removal of QM. These findings suggested that the C-block DNA of A. argenteus might undergo a structural change, such as strand breaks, during QM-BL processing. Application of the modified in situ nick-translation method revealed the generation of an appreciable number of nicks in the C-block DNA by QM-BL processing. No such nick formation was observed in the C-blocks of three other mammalian species: Apodemus peninsulae, Microtus montebelli, and Urotrichus talpoides. Our findings support the hypothesis that nick formation due to exposure to BL might play a primary role in inducing delayed QM-fluorescence in the C-blocks of A. argenteus. On the basis of the present and earlier findings, we propose a probable mechanism for delayed QM-fluorescence in A. argenteus chromosomes.